This project aims to elucidate the mechanisms of macrophage-mediated chemoprotection in breast cancer. We have found that in response to paclitaxel (Taxol) treatment there is an increase in tumor-associated macrophages (TAMs) and cathepsin protease levels in primary breast tumors in patients and in animal models. Cathepsin-expressing macrophages protected against Taxol-induced tumor cell death in co-culture, an effect fully reversed by cathepsin inhibition and mediated partially by cathepsins B and S. Macrophages were also found to protect against tumor cell death induced by additional chemotherapeutics, specifically etoposide and doxorubicin. Combining Taxol with cathepsin inhibition in vivo significantly enhanced efficacy against primary and metastatic tumors, supporting the therapeutic relevance of this effect. The main hypothesis of this application is that macrophage-secreted cathepsins directly modulate tumor cell survival signaling through their proteolytic activity, rapidly inducing a chemoresistant state. The objectives of this application are to identify the molecular mechanisms underlying microenvironmental chemoresistance adaptations, and develop therapeutic strategies to block chemoprotection in tumors. The first aim will be to determine the necessity and sufficiency of macrophage-secreted cathepsins to mediate protection against chemotherapy-induced tumor cell death. The second aim will be to identify the tumor cell survival signaling pathway(s) modulated by cathepsin activity, and determine the genomic alterations that dictate sensitivity to this mechanism of chemoprotection. The third aim will be to evaluate whether acute or enhanced cathepsin inhibitor treatment will result in a greater improvement in chemotherapeutic response in vivo, and determine the efficacy of adding cathepsin inhibitors to adjuvant treatment regimens. The implications of this work extend beyond the breast tumor microenvironment and through the elucidation of molecular mechanism we hope to identify other cancer types and organ sites where macrophages and cathepsin proteases contribute to treatment failure. We thereby intend to further demonstrate the critical importance of integrated therapeutic targeting of the tumor and its microenvironment.